Method and apparatus for improved detection of ionic species by capillary electrophoresis

ABSTRACT

Apparatus and methods are provided for increasing the sensitivity of detection of ionic species separated by capillary electrophoresis. The apparatus includes capillary electrophoretic separating means, suppressor means and detector means.

FIELD OF THE INVENTION

The present invention relates to method and apparatus using capillaryelectrophoresis and ion suppression for the determination of anions orcations.

BACKGROUND OF THE INVENTION

Capillary electrophoresis is a known technique involving electrophoresisin small bore capillaries. This approach provides methods for efficientanalytical separations of ionic species including macromolecules. Atypical capillary electrophoresis system is shown in FIG. 1. As can beseen, a capillary is positioned between two solvent reservoirscontaining electrolyte. Electrodes present in each of the reservoirs andcoupled to a power supply capable of delivering upwards to 30 kV per 100cm of capillary provide a voltage gradient to drive charged speciesthrough the capillary bore. A detector is positioned at a point betweenthe two high voltage electrodes to permit detection of various ionicspecies migrating in the capillary. A detector so positioned issometimes referred to as an on-column detector.

A number of approaches have been developed to detect the solutesseparated by capillary electrophoresis depending, in part, upon thenature of the solute detected. UV absorption and fluorescence have beenthe most commonly used detection modes. Mass spectrometric, radiometricand electrochemical methods of detection have also been utilized. Withregard to electrochemical detection, amperometry and conductivitymethods have been used.

Off-column amperometric detection utilizing a porous glass capillary tocover a crack in the capillary has also been reported. Wallingford, R.A., et al., Anal. Chem. (1987) 59, 1762-1766; Ewing, A. G., et al.,Anal. Chem. (1989) 61, 292A-303A. End-column amperometry andconductivity detection have also been performed. Huang, et al., Anal.chem. (1991), 63, 189-192. A significant problem with on-column andend-column detection utilizing electrochemical techniques is the effectof the high voltage applied to the electrodes to generate the voltagegradient across the capillary. Small variations in this high voltagegradient (typically ranging from 20-30 kV) have significant impact uponthe voltages used for amperometric detection and conductivity detectionwhich generally utilizes less than 1 volt in such detection systems.

Even for off-column electrochemical detection, a significant signal tonoise problem exists due to the presence of relatively highconcentrations of the electrolyte needed in the eluent to generate thevoltage gradient across the length of the capillary bore. This isespecially problematic for detection utilizing conductivity since suchmeasurements are more significantly affected by electrolyteconcentration than amperometric detection which is based primarily uponthe detection of redox reactions at the detection electrodes.

Although electrolyte suppression has been used primarily in ion exchangechromatography in conjunction with conductivity detection (see e.g. U.S.Pat. Nos. 3,897,213 3, 3,920,397, 3,925,019, 3,956,559, 4,474,664,4,751,004, 4,459,357 and 4,999,098), such suppressors have not beenadapted for use with capillary electrophoresis.

The references discussed above are provided solely for their disclosureprior to the filing date of the present application and nothing hereinis to be construed as an admission that the inventor is not entitled toantedate such disclosure by virtue of prior invention.

SUMMARY OF THE INVENTION

In accordance with the invention, apparatus and methods are provided forincreasing the sensitivity of detection of ionic species separated bycapillary electrophoresis. The apparatus includes capillaryelectrophoretic separating means, suppressor means and detector means.The capillary electrophoretic separator means typically include a smallbore capillary having a first end in communication with a firstelectrolyte reservoir which also contains a first electrode means. Thecapillary also has a second end having an ion conduction means incommunication with the second electrolyte reservoir containing a secondelectrode means. The first end of the capillary is capable of receivingelectrolyte from the first reservoir which is present throughout theentire bore of the capillary. The ion conducting means at the second endof the capillary is capable of conducting current generated between thefirst and second electrodes when an electrolyte is present in saidcapillary and said first and said second reservoirs and a voltage isapplied to the electrodes. This construction essentially isolates thesuppressor means and detector means from the current and high voltagegradient generated between the two electrodes.

The suppressor means comprises at least one capillary effluentcompartment having an inlet and an outlet end. The inlet end is incommunication with the second end of the capillary in the capillaryelectrophoretic separating means. The suppressor means also contains atleast one regenerant compartment means and at least one ion exchangemember partitioning the capillary effluent compartment means and theregenerant compartment means. This ion exchange membrane is permeable toions of the same charge (i.e., positive or negative) as the counter ionof the ionic species to be separated and impermeable to ions of the samecharge as the ionic species to be separated.

The detector means of the apparatus is in communication with the outletend of the suppressor means and is suitable for detecting resolved ionicspecies eluting therefrom.

In operation, the ionic species of the sample are separated in the boreof the capillary of the electrophoretic separating means by a voltagegradient generated by applying a voltage across the electrodes. Theeffluent from this separating means flows into the suppressor means andin particular the capillary effluent compartment means. The regenerantcompartment means contains a regenerant which is dissociated intocations and anions. Since the ion exchange membrane partitioning thecapillary effluent compartment and regenerant compartment is preferablypermeable to ions of the same charge (i.e., positive or negative) as thecounter ions of the ionic species to be detected, such counter ions arecapable of crossing the membrane into the regenerant compartment. Theregenerant ion of the same charge is capable of crossing the membranefrom the regenerant compartment into the effluent in the capillaryeffluent compartment. The net effect is to exchange the counter ion ofthe ionic species of interest.

For example, if an anion is to be detected and the electrolyte is sodiumhydroxide, a typical regenerant is sulfuric acid. For each molecule ofelectrolyte one sodium ion is replaced with a hydronium ion from theregenerant sulfuric acid to form water. Similarly, the sodium ionassociated with the anionic species of interest is replaced with ahydronium ion from the regenerant solution. As a consequence, theoverall conductivity of the eluent decreases and the overallconductivity of ionic species and counter ion increases. The thustreated effluent then flows to a detector. Since the overallconcentration of electrolyte has been reduced, the sensitivity ofdetecting conductivity increases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a prior art apparatus for performingcapillary electrophoresis derived from FIG. 1 of Ewing, et al., Anal.Chem. (1989), 61, 292A-303A.

FIG. 2 is a schematic diagram of one of the embodiments of theinvention.

FIG. 3 is a schematic diagram of the ion conducting means and suppressormeans used in Example 1.

FIG. 4 is an electropherogram demonstrating the separation and detectionof various anionic species by measuring conductivity using an embodimentof the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The system of the present invention is useful for determining a largenumber of solutes so long as such solutes are anions or cations. Asuitable sample includes surface waters and other liquids such asindustrial chemical wastes, body fluids, beverages such as fruits andwines and drinking water. When the term "ionic species" is used herein,it includes species in ionic form and components of molecules which areionizable under the conditions of the present system.

Generally, the ionic species which are particularly susceptible todetection by the apparatus and methods disclosed herein are thosespecies that absorb visible or ultraviolet light only weakly andtherefore are poorly detected by photometric absorbance detection.Examples of weakly absorbing species are common inorganic ions such aschloride, sulfate, sodium and potassium as well as many organic speciessuch as acetate, succinate and trimethylamine.

As used herein, a "capillary electrophoretic separating means" refers toany means for separating ionic species which contains an elongate narrowbore through which an electrolyte can be passed and which has ends thatcan be placed in contact with first and second electrolyte reservoirs.Although in the preferred embodiments, typical narrow bore capillariesgenerally used in prior art devices are used, the term "capillary" isnot limited to such capillaries. Rather, as used herein capillary refersto any elongate bore in a solid support having the dimensions on theorder of magnitude of the internal dimensions of prior art capillarydevices. Such capillaries have acceptable bore diameters ranging from 1to 1000μm, more preferably 25 to 100 μm. Generally, the length of suchcapillaries are about 1 cm to 10 meters, more preferably 20 cm to 100cm. Based on these parameters, capillaries for use in practicing thepresent invention may comprise such capillaries or may comprise channelsof irregular or regular shape formed in a solid support such as silicaby etching or machining. In some instances it may be desired to form oneside of a capillary in two separate blocks of solid support materialwhich can thereafter be joined to form the complete capillary. Ingeneral, the cross-sectional area of such non-standard capillaries issubstantially similar to that of conventional capillaries.

Referring to FIG. 2, a simplified apparatus for performing the presentinvention is illustrated. The system includes capillary 10 whichcontains a first end 12 immersed in an electrolyte reservoir 14. Alsocontained within this reservoir is first electrode 16 and an electrolyte18. This first electrode 16 is connected to high voltage supply unit 20.The second end of the capillary 10 has an ion conduction means 22attached thereto. This ion conduction means can be any conducting devicethat allows conduction of the current resulting from the high voltagecurrent generated between electrodes 16 and 24 through capillary 10.Such conducting means, because of the use of suppressor means anddetector downstream from the ion conduction means, requires that thision conduction means be chosen such that a substantial amount of themass flow through capillary 10 pass by ion conduction means 22 towardsuppressor tube 34. One embodiment of conductive means comprises a gapbetween the capillaries with an insulating sleeve having one or moreconducting holes. Alternatively, the insultaing sleeve may be looslyfitting to form an eluent gap between the capillaries and the sleeve. Afurther embodiment of the ion conduction means includes a capillary gapwith a porous sleeve capable of conducting ions through the porescontained therein. Such gaps, pores or holes are used to provide achannel for ionic current between the eluent at the second end ofcapillary lo and the second electrode 24 and should be chosen such thatat least about 50% of the mass flow passes such conducting means toremain in the downstream mass flow channel 26. In general, the nature ofsuch gaps, holes or pores should be large enough to provide sufficientionic current to complete the high voltage circuit but not be so largeas to allow the eluent to be diverted into reservoir 30.

In the preferred embodiments, the ion conductivity means 22 is anamphoteric membrane, preferably a sleeve which is capable of engagingthe second end of capillary 10 and an end of downstream conduit 28. Aspecific method for producing such an amphoteric membrane sleeve isdisclosed in Example 1. Other methods will be readily apparent to thoseskilled in the art and basically involve derivitizing a membrane withboth anionic and cationic functional groups.

The second reservoir 30 also contains an electrolyte 32 which is incontact with the second electrode 24 and the ionic conduction means 22to complete the high voltage circuit. As a consequence, the voltage dropalong the remaining flow path to the detector is negligible.

In general, the electrolyte present in at least the first reservoir andthe capillary bore is dependent upon the ionic species to be detected.When the ionic species is an anion, the electrolyte is preferably thesalt form of a weak acid. Examples include sodium borate, sodiumcarbonate and sodium hydorxide. When the ionic species to be detected isa cation, the electrolyte is preferably the salt form of a weak base. Anexample of such an electrolyte is hydrogen chloride.

An important aspect of practicing the invention, however, involves theproper choice of voltage to be applied across the electrophoresiselectrodes 16 and 24. Since a high voltage circuit is produced betweenelectrode 16 and electrode 24 via the ionic conducting means 22 andelectrolyte solution 32, there is no voltage potential downstream fromionic conducting means 22 to drive the ionic species and the electrolyteinto the suppressor means. To overcome this problem, the voltage ischosen such that it induces an electroendosmotic flow in the directionfrom the first reservoir to the detector. Electroendosmotic flow isgenerated primarily because of a local voltage which exists at theinterface between the inside surface of the capillary and theelectrolyte solution. This potential, sometimes referred to as the zetapotential, is dependent upon the material forming the inside surface ofthe capillary and the solution in contact with this surface.

In a silica capillary the charge on the inside wall of the capillary isnegative and the direction of electroendosmotic flow is toward thenegative electrode or away from the positive electrode. It is possibleto reverse the direction of electroendosmotic flow by coating orderivatizing the wall of the silica capillary such that the charge onthe wall is positive instead of negative. Under such circumstances toensure electroendosmotic flow to the detector, the polarity of theelectrode in the first reservoir should be reversed. It is also possibleto use capillaries made of materials such as polyethylene or polystyrenethat have no surface charge. These materials can then be functionalizedto generate a positive or negative charge for example by quanternizationor sulfonation respectively using known chemistries. The major reasonfor controlling electroendosmotic flow is to control separationresolution of the ionic species being separated. If theelectroendosmotic flow is the same direction as the electrophoreticdirection of the ionic species the separation resolution is less thanwhen the electroendosmotic flow is in the opposite direction of theelectrophoretic direction.

The downstream connector 28 is connected to a suppressor tube 34contained in a regenerant compartment 36. The regenerant compartment 36contains a regenerant 38 appropriate for the ionic species to bedetected and the electrolyte used for capillary electrophoresis.

The suppressor tube 34 has an internal bore which forms a compartment tocontain the effluent from the capillary 10. The inlet portion of thiscapillary effluent compartment is connected to downstream conduit means28. An ion exchange membrane is positioned between this capillaryeffluent and the regenerant compartment. In general, suppressor tube 34comprises an ion exchange membrane to facilitate ionic transport betweenthe capillary effluent compartment and the regenerant compartment. Theion exchange membrane is chosen such that it is preferentially permeableto the counter ion of the ionic species. Thus, if the ionic species isan anion its counter ion is a cation. The ion exchange membrane ischosen to be permeable to cations. The converse is also true. Suchtubular membranes can be purchased commercially such as the cationexchange membrane tube Nafion® available from Perma Pure Products, TomsRiver, N.J. It is to be understood, however, that the suppressor meansneed not be a tubular form of an ion exchange membrane. Alternateembodiments include flat membranes such as that disclosed in U.S. Pat.No. 4,999,098 incorporated herein by reference.

Although it is preferable that the cross-sectional area of thedownstream conduit 28 be the same as that of the cross-sectional area ofthe bore of capillary 10 and further that the cross-sectional area ofthe capillary effluent compartment 34 have substantially the samecross-sectional area, it is not necessary that such cross-sectionalareas be the same. Useful results have been obtained where the insidediameter of the circular bore capillary 10 was 75 microns and the insidediameter of the Nafion® cation exchange membrane tube was 400 microns.However, these results can be further improved when a 350 μm diameterplug is inserted inside the Nafion® tube to reduce the effectivecross-sectional flow area of the ion suppressor (see Example 1).

The suppressor length should be suffiecient to exchange cations oranions in the effluent but not be so long as to result in substantialband broadening. Such suppressor length can be from 100 μm to 10 cm,preferably 1 mm to 1 cm.

The foregoing describes a passive diffusion suppressor means forreducing electrolyte concentration. However, any suppressor system canbe used in practicing the invention including those utilizing theapplication of electrical fields across the ion exchange membrane toincrease the rate of ion exchange above that which would otherwise beobtained by diffusion limited exchange kinetics. See e.g. U.S. Pat. No.4,999,098 incorporated herein by reference.

After passing the ion exchange membrane, the thus treated effluent issubstantially depleted in the electrolyte and counter ions to the ionicspecies of interest. Such treated effluent may be directed to a flowthrough conductivity cell for detection of the ionic species present.Alternatively, one or both of the detection electrodes 42 and 44 can beinserted into the treated effluent stream exiting the suppressor means.When used in this manner, the outlet of the suppressor means may bemaintained in the regenerant solution 38 provided the electrodes arepositioned sufficiently within this end region so that conductivity isnot adversely influenced by diffusion of regenerant ions into theeffluent stream. In general, when one or more of the electrodes areinserted into the suppressor means outlet, the linear flow rate of theeffluent should be sufficient to offset the potential diffusion of theregenerant ions into the effluent stream which would otherwise defeatthe purpose of the suppressor means.

Although the foregoing has described the invention primarily within thecontext of open-tube capillary zone electrophoresis, it is believed thatother modes of capillary electrophoresis may be practiced using theinvention. Such other modes include isotachophoresis, micellarelectrokinetic capillary chromatography, capillary gel electrophoresisand isoelectric focusing.

The following sets forth a specific embodiment of the invention and isnot to be construed as a limitation of the claims as appended hereto.

EXAMPLE 1

Referring to FIG. 3, the separation capillary 10 was a fused silicacapillary with dimensions of 75 μm i.d., 375 μm o.d., and 75 cm length.It was butted up against a second fused silica capillary 28 of the samei.d. and o.d. but about 6 mm long. The amphoteric ion exchange membranetube 22 with dimensions of about 400 μm i.d., 800 μm o.d., and about 1cm long also acted as a sleeve to hold the two pieces of capillary 10and 28 together. The amphoteric membrane sleeve was attached to thecapillaries by tightly tying nylon monofilament (75 μm diameter) aroundthe sleeve. The amphoteric membrane sleeve was made from a substratetube made of polyethylene/polyvinyl acetate. This tube (Microline®) wasobtained from Thermal Plastic Scientific, Warren, N.J. It was radiationgrafted with vinylbenzylchloride (VBC) to about 50% VBC by weight. Thegrafted tube was refluxed for 14 hours in a 1M solution ofdimethylaminoacetonitrile in methanol and allowed to cool for 6 hours.Following rinses in methanol and water, the tube was heated to about 60°C. in 1M sodium hydroxide solution for 90 minutes. This reactionhydrolyzes the --CN to --CO₂ ⁻. The resulting tube contains thefollowing functional group covalently bonded to the VBC molecules in themembrane: -- N⁺ (CH₃)₂ CH₂ CO₂ ⁻.

The other end of capillary 28 was inserted into a length of Nafion®cation exchange tubing with approximate dimensions of 400 μm i.d., 800μm o.d., and 1 cm long (Perma Pure Products, Toms River, N.J.) whichserved as the suppressor 34. The suppressor was held onto capillary 28by tying with Nylon monofilament. Much of the internal volume of thesuppressor was taken up with a 350 μm o.d. by 5 mm long plug made fromcapillary with the ends sealed with epoxy resin. The approximate lengthof the suppressor from the exit of tube 28 to the detector electrode 44was 6 mm. One of the detector electrodes 44 was made by inserting a 127μm platinum wire into a 150 μm i.d., 350 μm o.d. by 2 cm long capillaryand filling the remaining volume with epoxy resin. The face of thecapillary was polished to expose the end of the platinum wire. Thiselectrode was inserted about 1 mm inside the suppressor, and the otherend of the wire was connected to the conductivity detector cables. Thesecond detector electrode 42 was placed into the regenerant solution 38.

The electrolyte in both the first electrolyte reservoir and in thesecond electrolyte reservoir was a 10 mM borax solution, pH 9.1. Theregenerant 38 was a 15 mM sulfuric acid solution.

The applied high-voltage was 20 kV, which resulted in a current of 20.5μA. A 5 second electrophoretic injection was used to inject a sample of20 μM each of the following anions: quinate, benzoate, benzenesulfonate,acetate, phthalate, phosphate, formate, and fluoride. The results areshown in FIG. 4.

Having described the preferred embodiments, it will be apparent to thoseskilled in the art that various modifications may be made to suchembodiments and that such modifications are intended to be within thescope of the invention.

What is claimed is:
 1. Apparatus for ion analysis comprising(a)capillary electrophoretic separating means for separating ionic speciescomprising a capillary having a first end in communication with a firstelectrolyte reservoir containing first electrode means and a second endhaving ion conduction means in communication with a second electrolytereservoir having second electrode means, said first end of saidcapillary being capable of receiving electrolyte from said firstreservoir and said ion conducting means at said second end of saidcapillary being capable of conducting the current generated between saidfirst and said second electrodes when an electrolyte is present in saidcapillary and said first and said second reservoirs and a voltage isapplied to said electrodes, (b) suppressor means for treating effluenteluted from said capillary electrophoretic separating means, saidsuppressor means including(1) at least one capillary effluentcompartment means having an inlet end and an outlet end, said inlet endbeing in communication with said second end of said capillary, (2) atleast one regenerant compartment means and (3) at least one ion exchangemembrane partitioning said capillary effluent compartment means and saidregenerant compartment means, said ion exchange membrane beingpreferentially permeable to the counter ion of said ionic species, and(c) detector means in communication with the outlet end of saidsuppressor means suitable for detecting resolved ionic species elutingtherefrom.
 2. The apparatus of claim 1 wherein said detector meanscomprise first and second spaced detection electrode means wherein atleast one of said detection electrode means is in electricalcommunication with the effluent from said outlet end of said suppressormeans.
 3. The apparatus of claim 2 wherein said detecting of saidresolved ionic species comprises measuring the conductivity of theeffluent from said outlet end of said suppressor means.
 4. The apparatusof claim 1 wherein said capillary effluent compartment and said ionexchange membrane in said suppressor means have substantially the samecross-sectional area.
 5. The apparatus of claim 4 wherein said capillaryand said selective ion exchange membrane tube have a circular crosssection of substantially the same diameter.
 6. The apparatus of claim 1wherein said ion conduction means comprises an amphoteric ion exchangemembrane.
 7. A method of ion analysis comprising(a) passing a samplecontaining ionic species in an electrolyte solution, includingtransmembrane electrolyte ions of opposite charge to said ionic species,through capillary electrophoretic separating means in which said ionicspecies are separated and wherein said capillary electrophoreticseparating means comprises a capillary having first and second endswherein said first end is in contact with a first electrolyte reservoircontaining electrolyte and first electrode means and said second endcomprises ionic conduction means in communication with a secondelectrolyte reservoir containing electrolyte and second electrode means,said first and said second electrode means having a voltage appliedthereto to cause the separation of said ionic species, (b) flowing theeffluent from the capillary electrophoretic separating means through asuppressor means having at least one capillary effluent compartmentmeans, at least one regenerant compartment means containing regenerantand at least one ion exchange membrane partitioning said capillaryeffluent and said regenerant compartment means, said ion exchangemembrane being preferentially permeable to the counter ion of said ionicspecies to allow said counter ion to cross said membrane into saidregenerant compartment means and regenerant ions of the same charge topass said membrane into said effluent, and (c) flowing the treatedeffluent from the outlet of said capillary effluent compartment in saidsuppressor means through detection means in which said separated ionicspecies are detected by measuring the conductivity of said treatedeffluent.
 8. The method of claim 7 wherein the voltage applied to saidfirst and said second electrode means is sufficient to induceelectroendosmotic flow through said capillary.
 9. The method of claim 8wherein said electroendosmotic flow is from said first end to saidsecond end of said capillary, the internal surface of said capillary hasa negative charge and said first electrode has a positive polarity ascompared to said second electrode.
 10. The method of claim 8 whereinsaid electroendosmotic flow is from said first end to said second end ofsaid capillary, the internal surface of said capillary has a positivecharge and said first electrode has a negative polarity as compared tosaid second electrode.